Many physiological processes function efficiently within a well-controlled pH range and significant deviations from this range typically are indicative of pathologic conditions. Within cells, maintenance of a pH gradient in different organelles allows for differential activation of certain enzymes or generation of active conformations of proteins for transient or prolonged activity. Globally, the sum of cellular events can reflect on the pH of tissues and organ systems. A common strategy to monitor the pH of biochemical events relies on the use of pH-sensitive fluorescent molecular probes to monitor diverse physiological and pathological processes, including studies related to cancer, cell proliferation, endocytotic and other physiological processes. Currently, there is a major disconnection between the use of pH-sensitive fluorescent dyes in molecular cell biology and for imaging diseases in vivo. This is because most of these dyes fluoresce in the visible wavelengths where absorption of light by native fluorophores and autofluorescence limits their use in both tissue and cell imaging. To overcome these problems, we propose to develop new classes of pH-sensitive near infrared fluorescent molecular probes that are useful for imaging molecular processes by microscopy and whole-body animal imaging by diffuse optical tomography. Advantages of this approach include high detection sensitivity because of low autofluorescence, low phototoxicity because of the use of low energy radiation, and the extension of the spectral window to the 900 nm which allows the use of multiple fluorophores between 400 and 900 nm wavelengths. More importantly, information gleaned from the molecular cell biology studies can be translated to small animals, and eventually to humans with the same pHsensitive NIR molecular probe because NIR light can penetrate into deep tissues. We will accomplish these goals by (1) designing and developing two new classes of pHsensitive NIR dyes;(2) demonstrating their utility to image pH-dependent molecular processes in cells;and (3) imaging the onset of acidosis in solid tumors in vivo. The products generated from this project will be distributed to other investigators for research purposes. Project Narrative The goals of this project are to (1) design and develop two new classes of pH-sensitive NIR dyes;(2) demonstrate their utility to image pH-dependent molecular processes in cells by microscopy;and (3) image the onset of acidosis in solid tumors in vivo. The technology platform developed will be useful for diverse pH-based biological applications.